The development of gene therapy for hemophilia and other diseases remains stymied by a failure to achieve clinically significant levels of transgene integration in targeted cells. Adeno-associated virus integrates into AAVS1, a site on Human Chromosome 19qter (AAVS1). Increasing evidence indicates the involvement of both the viral Rep proteins and the AAV Inverted Terminal Repeats (ITR) in this integration process. The goal of this project is to utilize AAV functions in order to develop non-viral vectors that will result in the targeted integration of heterologous transgenes, including the genes for Human Coagulation Factors VIII and IX. Results: (1) In non-clonal Hepatoma G-2 cells, codelivery of the Rep protein by lipid-based transfection resulted in a long-term, Rep dose-dependent increase in the expression of a marker gene flanked by AAV ITRs. (2) In 293 cells the presence of both Rep (delivered by liposomes as either a protein or as a gene) and AAV ITRs increased the frequency of clonal cells with longterm transgene expression. (3) In order to develop an in vivo model for understanding AAV integration, a mouse transgenic for AAVS1 was created. (4) These mice appear to develop normally without any evidence of pathology. (5) AAVS1 transgenic mice were bred to homozygosity. (4) We are presently evaluating these animals using viral and non-viral AAV vectors in which AAV Rep functions are provided in trans.